Tyrosine kinases are critical mediators of intracellular signaling that are misregulated in numerous cancers. Tyrosine kinase inhibitors are therefore emerging as important therapeutics in oncology. In addition, tyrosine kinase signaling is broadly implicated as important in other diseases, including diabetes, heart disease, and viral and bacterial infectivity. The importance of tyrosine phosphorylation in disease has inspired the development of numerous approaches to interrogate tyrosine kinase-mediated signaling.
Tyrosine phosphatases have critical roles in the temporal dynamics of cell signaling via their role in dephosphorylating kinases and kinase substrates. Changes in tyrosine phosphatase activities are associated with numerous human diseases, including diabetes, obesity, inflammatory diseases, and some cancers. Accordingly, inhibitors of tyrosine phosphatases have received increasing attention as possible therapeutics.
Several groups have developed encodable sensor systems for tyrosine kinases Src, Abl, and the EGF receptor. (Ting et al., 2001, Proc. Natl. Acad. Sci. USA 98:15003-15008.) These systems are based on changes in fluorescence resonance energy transfer and involve four components: cyan fluorescent protein, a kinase recognition site, a binding module for the phosphorylated (kinase-activated) protein, and yellow fluorescent protein. These systems appear to be reasonably generally applicable to different protein kinases if a binding module is known or can be developed for a kinase recognition site. However, the changes in fluorescence on phosphorylation are relatively modest for these systems, reducing sensitivity to small changes. In addition, the large size (greater than 50 kD) of the structures limits the potential applications of the system, particularly their employment as protein tags, (1) due to the possibility of steric interference by the sensor system if it were used as a protein tag; (2) by making full biophysical characterization of the system difficult; and (3) by increasing the development time for new kinase sensors. These encodable systems are in general not well suited to solution assays due to their size.
Sensitive fluorescence-based sensing systems have been developed for protein kinase activity, demonstrating as much as 9-fold fluorescence difference between non-phosphorylated and phosphorylated protein. (Lawrence and Wang, 2007, ChemBioChem 8:373-378; Wang and Lawrence, 2005, Am. Chem. Soc. 127:7684-7685; Wang et al., 2006, J. Am. Chem. Soc. 128:1808-1809,) These probes can allow real-time analysis of kinase activity and thus hold great promise, and are clearly far more convenient than previously employed radioassays or immunoassays of protein kinase activity. However, these sensors have the limitation that they incorporate external fluorophores and thus cannot be expressed. Similarly, kinase probes have been demonstrated for the activities of multiple kinases having as much as 10-fold differences in fluorescence between phosphorylated and non-phosphorylated peptide. (Shults and Imperiali, 2003, J. Am. Chem. Soc. 125:14248-14249; Vazquez et al., 2003, J. Am. Chem. Soc. 125:10150-10151; Shults et al., 2005, Nature Methods 2:277-283.) However, these systems are based on an unnatural amino acid and thus also cannot be expressed in vivo. In addition, the unnatural amino acids employed are not commercially available. Moreover, in most cases these systems are generally limited to kinase recognition sequences that are either N-terminal or C-terminal to the tyrosine residue that is phosphorylated. However, the specificity in protein phosphorylation by protein kinases usually involves residues both N-terminal and C-terminal to the phosphorylated residue. In addition, in many signaling cascades, kinase specificity is dependent on scaffolding proteins and protein-binding domains, requiring the localization of a sensor as a tag (ideally small) on substrate proteins. While synthetic constructs have been developed combining recognition sequences and a targeting protein, the absence of encodability limits the applications of these systems. (Placzek et al., 2010, Analytical Biochem. 397:73-78; Lukovic et al., 2009, Angewandte Chemie-International Edition 48:6828-6831.)
A protein kinase-inducible domain (pKID) was previously designed to become structured and bind lanthanides when phosphorylated on serine or threonine. (Balakrishnan and Zondlo, 2006, J. Am. Chem. Soc. 128:5590-5591; U.S. Pat. No. 7,816,102.) In that design, phosphoserine or phosphothreonine mimicked a structurally important Glu residue, generating phosphorylation-dependent EF-hand peptides. In the design of a protein dependent on tyrosine phosphorylation, the size differences between phosphotyrosine and Glu precludes an approach involving direct mimicry of Glu by phosphotyrosine.
To date, no system combines large changes in fluorescence with the ability to express the kinase sensor as a protein tag. Given the importance of tyrosine kinase and phosphatase activities in human health and diseases, there remains a need for new tools to assess changes in tyrosine kinase and/or phosphatase activity as a function of a cell state or disease.